The neurosteroid allopregnanolone (ALLO) is a potent positive modulator of GABAA receptors, and evidence suggests that GABAergic neurosteroids are endogenous modulators of GABAA receptors and of selective effects of ethanol (EtOH). The present proposal builds on results generated in the current period of funding in the selectively bred Withdrawal Seizure-Prone (WSP) and -Resistant (WSR) male mice. The finding that WSP mice had a persistent decrease in endogenous ALLO levels during EtOH withdrawal, in conjunction with tolerance to ALLO's anticonvulsant effect, is consistent with greater neural excitability in the WSP vs. WSR line during EtOH withdrawal. The selected line differences in the modulatory effect of ALLO on EtOH withdrawal severity likely reflects a balance between alterations in local concentration of ALLO at GABAA receptors and the concomitant change in GABAA receptor sensitivity to ALLO during EtOH withdrawal. Thus, the goals of the proposed studies are to determine the mechanism and site(s) of action underlying the tolerance to ALLO during EtOH withdrawal in WSP mice (Aim 3), the relative contribution of altered local endogenous ALLO levels (Aim 2) and altered expression of GABAA receptor subunits (Aim 4) to the line difference in ALLO sensitivity during EtOH withdrawal severity in WSP and WSR mice, and the anatomical localization and regulation of the biosynthetic enzyme 5a-reductase (Srd5a1) during EtOH withdrawal in WSP and WSR mice (Aim 1). The pattern of the results will provide essential information on whether activation of GABAA receptors in particular brain regions will be sufficient to alter EtOH withdrawal severity or sensitivity to ALLO during EtOH withdrawal as well as the critical involvement of local ALLO concentration in a specific brain region on EtOH withdrawal severity. This multidisciplinary approach will further test the hypothesis that a decrease in endogenous ALLO levels, which alters GABAergic tone, in conjunction with a decrease in GABAA receptor sensitivity, contribute to the increased withdrawal severity in WSP mice. The long-term goal of this research is to understand mechanisms underlying a genetic predisposition for increased withdrawal severity. This information will aid in our understanding of the mechanisms underlying alcohol withdrawal and will help in the development of new strategies for the treatment of alcohol dependence.